Transient overexpression of human H- and L-ferritin chains in COS cells.
The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.
Corsi
B
B
Dibit, Institute H.San Raffaele, Via Olgettina 58, 20132 Milan, Italy.
Perrone
F
F
Bourgeois
M
M
Beaumont
C
C
Panzeri
M C
MC
Cozzi
A
A
Sangregorio
R
R
Santambrogio
P
P
Albertini
A
A
Arosio
P
P
Levi
S
S
D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism; D046911:Macromolecular Substances; D011108:Polymers; D011485:Protein Binding; D011994:Recombinant Proteins
D046911:Macromolecular Substances; D011108:Polymers; D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron